Cell adhesion is involved in a number of critical cellular processes including anchorage to the extracellular matrix and to other cells, growth, differentiation and migration. Cell adhesion is mediated by dimeric transmembrane receptor proteins called integrins (Ruoslahti, (1991) J. Clin. Invest., 87:1-5; Hynes, (1992) Cell, 69:11-25). Integrins promote cell adhesion to the extracellular matrix, a filamentous network of proteins secreted by cells, by binding to target sequences present in these proteins. The major target sequence recognized by integrins is an arginine-glycine-aspartate (RGD) motif present in numerous substrate proteins including fibronectin, vitronectin and laminin (Hemler, (1990) Annu. Rev. Immunol., 8:365-400). First identified in fibronectin, RGD has since been shown to be the cellular recognition sequence in many matrix proteins. Other sequence motifs have also been found to promote cell adhesion, including KQAGD found in fibrinogen and PDSGR found in laminin.
TGF-.beta. encompasses a family of dimeric proteins including TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5 which regulate the growth and differentiation of many cell types (Barnard et al., (1990) Biochim. Biophys. Acta., 1032:79-87). Other members of this family include the more distantly related Mullerian inhibitory substance (Cate et al., (1986) Cell, 45:685-698) and the inhibins (Mason et al., (1985) Nature, 318:659-663). TGF exhibits a diverse range of biological effects, stimulating the growth of some cell types (Noda et al., (1989) Endocrinology, 124:2991-2995) and inhibiting the growth of other cell types (Goey et al., (1989) J. Immunol., 143:877-880; Pietenpol et al., (1990) Proc. Natl. Acad. Sci. USA, 87:3758-3762). In regard to cell adhesion, TGF-.beta. increases the expression of collagen and fibronectin (Ignotz et al., (1986) J. Biol. Chem., 261:4337-4345) and accelerates the healing of incisional wounds (Mustoe et al., (1987) Science, 237:1333-1335).
Skonier et al. (DNA Cell Biol., 11:511-522, 1992) cloned and sequenced a TGF-.beta.1-induced gene isolated from a human lung adenocarcinoma cell line by constructing cDNA libraries from both TGF-.beta.1-stimulated and unstimulated cells and screening the libraries by subtractive hybridization. This gene encoded a 683 amino acid protein called .beta.IG-H3 (H3) which contained a carboxy-terminal RGD sequence. The protein also contained four internal repeats with limited homology to Drosophila fasciclin I, an extrinsic membrane protein thought to be involved in growth cone guidance, and a PDSAK sequence similar to the PDSGR active binding domain of laminin. The presence of these sequence motifs indicated that H3 could be involved in cell adhesion.
Numerous attempts have been made at increasing fibroblast adhesion to substrates. The main approach has involved the use of RGD-containing peptides (Quaglino, Jr., et al., (1991) J. Invest. Dermatol., 97:34-42; Peptide Res., 5:331-335; Agrez et al., (1991) Cell Regul., 2:1035-1044), although this method has had limited success. U.S. Pat. No. 4,963,489 to Naughton et al., the contents of which are hereby incorporated by reference, discloses a three-dimensional matrix and its use as a framework for a multi-layer cell culture system for the production of a number of cells and/or tissues by culturing desired cell types on a stromal cell layer.
There are currently no simple, effective methods for stimulating cell spreading and adhesion at wound sites to promote rapid wound healing. Thus, there is a need for substances able to promote attachment and spreading of cells, particularly fibroblasts, to facilitate this important process. Such a substance and its use in wound healing and tissue engineering applications are described herein.